The regulation of cyclic nucleotide phsophodiesterase and adenylate cyclase activities by Ca 2 ion will be investigated in cell free preparations of rat or beef brain and other tissues. Specific forms of each of these enzymes are deactivated by the removal on anion exchange chromatography of a Ca 2 ion-dependent regulator (CDR) identified previously as a heat stable, acidic Ca 2 ion-binding protein which has been purified to homogeneity. These enzymes are reactivated by the readdition of CDR and Ca 2 ion. Primary emphasis will be placed on characterizing a factor which dissociates from the adenylate cyclase and which is required for Ca 2 ion. CDR-dependent activation of the enzyme. This factor, which may be a CDR binding protein, will be purified and characterized physically, as will the deactivated catalytic unit of the enzyme. Antibodies will be raised to the factor. The interactions of Ca 2 ion. CDR, the separable factor, and the catalytic unit of the adenylate cyclase will be investigated by analyses of reaction kinetics, sucrose density gradient centrifugation studies, and direct binding studies. The tissue distribution of the factor and the CDR-dependent adenylate cyclase will be determined. The possibility that the factor also confers CDR dependence to the phosphodiesterase will be investigated. The relative Ca 2 ion concentration dependence of the adenylate cyclase and the phosphodiesterase will be determined as a function of CDR concentration, utilizing reagents freed of Ca 2 ion by chromatography on Chelex-100 as monitored by atomic absorption spectrophotometry, and compared with the concentration dependence of these enzymes and of a Ca 2 ion inhibitable form of phosphodiesterase. The uptake and binding of the CDR to membrane fractions prepared from brain homogenates by differential and discontinuous sucrose density gradients centrifugation will be studied.